Purpose

Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes.

Methods

Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70–80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein.

Results

Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a −16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein.

Conclusions

AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

中文翻译：

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人和兔睑板腺和睑板细胞表达酰基辅酶A蜡醇酰基转移酶2 (AWAT2)

目的

此前，我们发现，培养的永生化人睑板腺上皮细胞（hMGEC）不表达酰基辅酶A蜡醇酰基转移酶2（AWAT2），这是睑脂蜡酯合成所需的必需酶。为了开始了解控制 AWAT2 表达的机制，我们分析了其在人和兔睑板腺和培养的睑板细胞中的表达。

方法

兔睑板腺祖细胞 (rMPC) 首先在补充有 rhEGF、FGF10 和 ROCK 抑制剂（Y-27632 二盐酸盐）的 Cnt-BM.1 基础培养基 (Cellntec) 中生长，然后通过 Accutase 以 70-80% 汇合度通过。通过去除 Y-27632 并添加 1 mM 钙（含或不含 PPARγ 激动剂）诱导 rMPC 向睑板细胞 (rMC) 分化。从组织、原代、传代 rMPC 和分化 rMC 中获取 RNA 用于 AWAT2 qPCR 分析。分别评估蛋白质和细胞的蛋白质印迹和中性脂质合成。为了进行比较，分离人类睑板腺进行 RNA 和蛋白质分析。培养hMGEC以收集RNA和蛋白质。

结果

兔 rMPC 成功生长、传代和分化，显示脂滴积累显着增加。AWAT2 RNA 在组织中高表达，但在原代和传代 rMPC 中显示 -16.9 log2 倍下降，并且不被分化为 rMC 诱导。相比之下，人类睑板腺表现出 AWAT2 的高表达，而 hMGEC 表达的 AWAT2 转录物或蛋白质水平不可检测。

结论

AWAT2 表达在培养的 rMPC 和 rMC 中丢失，表明培养的细胞没有经历完全的睑母细胞分化，并且需要尚未确定的培养条件。